Pharmacological stimulation of fetal hemoglobin (Hb F) has been attracting great concerns for many years, but the molecular mechanisms by which expression of the g-globin gene is induced in the adult stage still remain unclear. The long-term goal of this proposal is to develop "novel Hb F inducers" by clarifying intracellular pathways that regulate g-globin gene expression. We showed that expression of the g-globin gene is induced in erythroleukemic cells as well as in primary erythroblasts by activating an intracellular pathway comprising soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). This pathway was also found to be essential for the induced expression of the g-globin gene by hemin or butyrate. In the first specific aim, we will study the molecular mechanisms by which the sGC-PKG pathway induces g-globin gene expression. First, we will identify and characterize within the b-globin locus cis-acting and trans-acting elements that mediate molecular effects of the pathway to the g-globin gene. Next, we will examine whether the sGC-PKG pathway contributes to the expression of the gamma-globin gene in beta-thalassemia. In the second specific aim, we will test using transgenic mice the hypothesis that expression of the g-globin gene is induced in the adult stage by over- expressing or activating sGC, which is an obligate heterodimer of a- and b- subunits. We will first create transgenic mice with DNA constructs carrying sGC subunit genes driven by the b-globin gene promoter and the LCR, and breed them with mice carrying the human b-globin locus. Second, we will examine whether the phenotype of sickle cell mice can be alleviated by expressing sGC subunits at high levels. Recently we found two normal subjects with no mutations in the b-globin locus who express 30% Hb F and have high levels of porphyrins such as protoporphyrin IX (PPIX) and ZnPP, both of which are sGC activators. Third, we will examine whether g-globin gene expression can be induced by increasing PPIX concentrations in red cells. This will be performed by breeding mice carrying the human b-globin locus with those of ferrochelatase deficiency. If successfully implemented, this proposal should not only enhance our understanding of the molecular mechanisms that regulate the expression of the g-globin gene during development, but also provide important information to develop novel Hb F inducers for treating the b-globin disorders.